This application addresses broad Challenge Area (15) Translational Science and specific Challenge Topic 15-RR-101 Applied Translational Technology Development. Smiths Detection Diagnostics, in collaboration with the UC Davis-LLNL Point-of-Care (POC) Technologies Center, one of four Centers in the NIBIB POC Technologies Research Network, and Brandeis University propose to develop a highly multiplexed LATE-PCR in vitro diagnostic assay for the identification of pathogens commonly associated with burn/wound infections and septicemia in a point-of-care (POC) setting. The proposed project will focus on technology development and technology translation of the Clinical Bio-SeeqTM System, a "sample in, answer out" POC PCR platform that utilizes an automated sample preparation cartridge and Linear After The Exponential PCR (LATE-PCR), a novel asymmetric PCR technique that provides significant performance benefits over real time PCR. The long-term goal of this project is to facilitate the translation of the Clinical Bio-SeeqTM System from its current advanced prototype status into the clinic, providing a platform which enables POC PCR testing capability for a broad range of clinical settings with a diverse menu of infectious disease assays available for use. The immediate objective of this project is to develop a highly multiplexed LATE-PCR assay for the identification of eight microbial pathogens commonly associated with burn/wound infections in the ICU and to demonstrate the feasibility of using this assay in a POC setting. Multiplex assay development will consist of bioinformatics analysis of genetic targets, design and optimization of single-target assays, and the sequential addition/optimization of these single- target assays into the multiplex assay. The assay design and optimization will take place in the Wangh Laboratory at Brandeis University. The multiplex assay will be validated for specificity against pure culture isolates at the UC Davis-LLNL Point-of-Care (POC) Technologies Center. Smiths Detection Diagnostics will perform the product development, creating lyophilized reagents and integrating the assay into the Clinical Bio-SeeqTM System consumables. The POC Technologies Center will then use the integrated multiplex assay and Clinical Bio-SeeqTM System to test clinical samples from ICU patients suspected of having a wound infection or sepsis and the results will be compared to replicate samples tested with standard laboratory methods used for infectious disease testing. PUBLIC HEALTH RELEVANCE: Sepsis is a major cause of mortality in non-coronary intensive care units. High risk, resistant pathogens such as MRSA are no longer confined to ICU's, acute care hospitals, or any healthcare institution, and therefore, have become a major public health and legal concern in the United States. LATE-PCR-based detection of high risk pathogens at the point-of-care will improve patient outcomes by identifying sepsis-related bacteria more rapidly, thereby allowing early and directed treatment.